Purified TNFR preparations

ABSTRACT

Purified protein preparations comprising tumor necrosis factor are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.11/260,192 (Conf. No. 7372), filed Oct. 28, 2005; which is a DivisionalApplication of pending prior application Ser. No. 10/420,785 (Conf. No.4626), filed Apr. 23, 2003 (now U.S. Pat. No. 7,057,022); which is aDivisional of U.S. application Ser. No. 09/758,124, filed Jan. 12, 2001(now U.S. Pat. No. 6,572,852); which is a Divisional of U.S. applicationSer. No. 08/953,268, filed Oct. 17, 1997 (now abandoned); which is aDivisional of U.S. application Ser. No. 08/555,629, filed Nov. 9, 1995(now abandoned); which is a Divisional of U.S. application Ser. No.08/468,453, filed Jun. 6, 1995 (now abandoned); which is a Continuationof U.S. application Ser. No. 08/038,765, filed Mar. 19, 1993 (nowabandoned); which is a Divisional of U.S. application Ser. No.07/523,635, filed May 10, 1990 (now U.S. Pat. No. 5,395,760); which is aContinuation-In-Part of U.S. application Ser. No. 07/421,417, filed Oct.13, 1989 (now abandoned); which is a Continuation-In-Part of U.S.application Ser. No. 07/405,370, filed Sep. 11, 1989 (now abandoned);which is a Continuation-In-Part of U.S. application Ser. No. 07/403,241,filed Sep. 5, 1989 (now abandoned). The entire disclosures of each ofthe prior applications is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to cytokine receptors and morespecifically to tumor necrosis factor receptors.

Tumor necrosis factor-α (TNFα, also known as cachectin) and tumornecrosis factor-β (TNFβ, also known as lymphotoxin) are homologousmammalian endogenous secretory proteins capable of inducing a widevariety of effects on a large number of cell types. The greatsimilarities in the structural and functional characteristics of thesetwo cytokines have resulted in their collective description as “TNF.”Complementary cDNA clones encoding TNFα (Pennica et al., Nature 312:724, 1984) and TNFβ (Gray et al., Nature 312: 721, 1984) have beenisolated, permitting further structural and biological characterizationof TNF.

TNF proteins initiate their biological effect on cells by binding tospecific TNF receptor (TNF-R) proteins expressed on the plasma membraneof a TNF-responsive cell. TNFα and TNFβ were first shown to bind to acommon receptor on the human cervical carcinoma cell line ME-180(Aggarwal et al., Nature 318: 665, 1985). Estimates of the size of theTNF-R determined by affinity labeling studies ranged from 54 to 175 kDa(Creasey et al, Proc. Natl. Acad Sci. USA 84: 3293, 1987; Stauber etal., J. Biol. Chem. 263: 19098, 1988; Hohmann et al., J. Biol. Chem.264: 14927, 1989). Although the relationship between these TNF-Rs ofdifferent molecular mass is unclear, Hohmann et al. (J. Biol. Chem. 264:14927, 1989) reported that at least two different cell surface receptorsfor TNF exist on different cell types. These receptors have an apparentmolecular mass of about 80 kDa and about 55-60 kDa, respectively. Noneof the above publications, however, reported the purification tohomogeneity of cell surface TNF receptors.

In addition to cell surface receptors for TNF, soluble proteins fromhuman urine capable of binding TNF have also been identified (Peetre etal., Eur. J. Haematol. 41: 414, 1988; Seckinger et al., J. Exp. Med.167: 1511, 1988; Seckinger et al., J. Biol. Chem. 264: 11966, 1989; UKPatent Application, Publ. No. 2 218 101 A to Seckinger et al.; Engelmannet al., J. Biol. Chem. 264: 11974, 1989). The soluble urinary TNFbinding protein disclosed by UK 2 218 101 A has a partial N-terminalamino acid sequence of Asp-Ser-Val-Cys-Pro-, which corresponds to thepartial sequence disclosed later by Engelmann et al. (1989). Therelationship of the above soluble urinary binding proteins was furtherelucidated after original parent application (U.S. Ser. No. 403,241) ofthe present application was filed, when Engelmann et al. reported theidentification and purification of a second distinct soluble urinary TNFbinding protein having an N-terminal amino acid sequence ofVal-Ala-Phe-Thr-Pro- (J. Biol. Chem. 265: 1531, 1990). The two urinaryproteins disclosed by the UK 2 218 101 A and the Engelmann et al.publications were shown to be immunochemically related to two apparentlydistinct cell surface proteins by the ability of antiserum against thebinding proteins to inhibit TNF binding to certain cells.

More recently, two separate groups reported the molecular cloning andexpression of a human 55 kDa TNF-R (Loetscher et al., Cell 61: 351,1990; Schall et al., Cell 61: 361, 1990). The TNF-R of both groups hasan N-terminal amino acid sequence which corresponds to the partial aminoacid sequence of the urinary binding protein disclosed. by UK 2 218 101A, Engelmann et al. (1989) and Englelmann et al. (1990).

In order to elucidate the relationship of the multiple forms of TNF-Rand soluble urinary TNF binding proteins, or to study the structural andbiological characteristics of TNF-Rs and the role played by TNF-Rs inthe responses of various cell populations to TNF or other cytokinestimulation, or to use TNF-Rs effectively in therapy, diagnosis, orassay, purified compositions of TNF-R are needed. Such compositions,however, are obtainable in practical yields only by cloning andexpressing genes encoding the receptors using recombinant DNAtechnology. Efforts to purify the TNF-R molecule for use in biochemicalanalysis or to clone and express mammalian genes encoding TNF-R,however, have been impeded by lack of a suitable source of receptorprotein or mRNA. Prior to the present invention, no cell lines wereknown to express high levels of TNF-R constitutively and continuously,which precluded purification of receptor for sequencing or constructionof genetic libraries for cDNA cloning.

SUMMARY OF THE INVENTION

The present invention provides isolated TNF receptors and DNA sequencesencoding mammalian tumor necrosis factor receptors (TNF-R), inparticular, human TNF-Rs. Such DNA sequences include (a) cDNA cloneshaving a nucleotide sequence derived from the coding region of a nativeTNF-R gene; (b) DNA sequences which are capable of hybridization to thecDNA clones of (a) under moderately stringent conditions and whichencode biologically active TNF-R molecules; or (c) DNA sequences whichare degenerate as a result of the genetic code to the DNA sequencesdefined in (a) and (b) and which encode biologically active TNF-Rmolecules. In particular, the present invention provides DNA sequenceswhich encode soluble TNF receptors.

The present invention also provides recombinant expression vectorscomprising the DNA sequences defined above, recombinant TNF-R moleculesproduced using the recombinant expression vectors, and processes forproducing the recombinant TNF-R molecules using the expression vectors.

The present invention also provides isolated or purified proteincompositions comprising TNF-R, and, in particular, soluble forms ofTNF-R.

The present invention also provides compositions for use in therapy,diagnosis, assay of TNF-R, or in raising antibodies to TNF-R, comprisingeffective quantities of soluble native or recombinant receptor proteinsprepared according to the foregoing processes.

Because of the ability of TNF to specifically bind TNF receptors(TNF-Rs), purified TNF-R compositions will be useful in diagnosticassays for TNF, as well as in raising antibodies to TNF receptor for usein diagnosis and therapy. In addition, purified TNF receptorcompositions may be used directly in therapy to bind or scavenge TNF,thereby providing a means for regulating the immune activities of thiscytokine.

These and other aspects of the present invention will become evidentupon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the coding region of variouscDNAs encoding human and murine TNF-Rs. The leader sequence is hatchedand the transmembrane region is solid.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “TNF receptor” and “TNF-R” refer to proteinshaving amino acid sequences which are substantially similar to thenative mammalian TNF receptor amino acid sequences, and which arebiologically active, as defined below, in that they are capable ofbinding TNF molecules or transducing a biological signal initiated by aTNF molecule binding to a cell, or cross-reacting with anti-TNF-Rantibodies raised against TNF-R from natural (i.e., nonrecombinant)sources. The mature full-length human TNF-R is a glycoprotein having amolecular weight of about 80 kilodaltons (kDa). As used throughout thespecification, the term “mature” means a protein expressed in a formlacking a leader sequence as may be present in full-length transcriptsof a native gene. Experiments using COS cells transfected with a cDNAencoding full-length human TNF-R showed that TNF-R bound ¹²⁵I-TNFα withan apparent Ka of about 5×10⁹ M⁻¹, and that TNF-R bound ¹²⁵I-TNFβ withan apparent Ka Of about 2×10⁹ M⁻¹. The terms “TNF receptor” or “TNF-R”include, but are not limited to, analogs or subunits of native proteinshaving at least 20 amino acids and which exhibit at least somebiological activity in common with TNF-R, for example, soluble TNF-Rconstructs which are devoid of a transmembrane region (and are secretedfrom the cell) but retain the ability to bind TNF. Various bioequivalentprotein and amino acid analogs are described in detail below.

The nomenclature for TNF-R analogs as used herein follows the conventionof naming the protein (e.g., TNF-R) preceded by either hu (for human) ormu (for murine) and followed by a Δ (to designate a deletion) and thenumber of the C-terminal amino acid. For example, huTNF-RΔ235 refers tohuman TNF-R having Asp²³⁵ as the C-terminal amino acid (i.e., apolypeptide having the sequence of amino acids 1-235 of SEQ ID NO:1). Inthe absence of any human or murine species designation, TNF-R refersgenerically to mammalian TNF-R. Similarly, in the absence of anyspecific designation for deletion mutants, the term TNF-R means allforms of TNF-R, including mutants and analogs which possess TNF-Rbiological activity.

“Soluble TNF-R” or “sTNF-R” as used in the context of the presentinvention refer to proteins, or substantially equivalent analogs, havingan amino acid sequence corresponding to all or part of the extracellularregion of a native TNF-R, for example, huTNF-RΔ235, huTNF-RΔ185 andhuTNF-RΔ163, or amino acid sequences substantially similar to thesequences of amino acids 1-163, amino acids 1-185, or amino acids 1-235of SEQ ID NO:1, and which are biologically active in that they bind toTNF ligand. Equivalent soluble TNF-Rs include polypeptides which varyfrom these sequences by one or more substitutions, deletions, oradditions, and which retain the ability to bind TNF or inhibit TNFsignal transduction activity via cell surface bound TNF receptorproteins, for example huTNF-RΔx, wherein x is selected from the groupconsisting of any one of amino acids 163-235 of SEQ ID NO:1. Analogousdeletions may be made to muTNF-R. Inhibition of TNF signal transductionactivity can be determined by transfecting cells with recombinant TNF-RDNAs to obtain recombinant receptor expression. The cells are thencontacted with TNF and the resulting metabolic effects examined. If aneffect results which is attributable to the action of the ligand, thenthe recombinant receptor has signal transduction activity. Exemplaryprocedures for determining whether a polypeptide has signal transductionactivity are disclosed by Idzerda et al., J. Exp. Med 171: 861 (1990);Curtis et al., Proc. Natl. Acad Sci. USA 86: 3045 (1989); Prywes et al.,EMBO J. 5: 2179 (1986) and Chou et al., J. Biol. Chem. 262: 1842 (1987).Alternatively, primary cells or cell lines which express an endogenousTNF receptor and have a detectable biological response to TNF could alsobe utilized.

The term “isolated” or “purified”, as used in the context of thisspecification to define the purity of TNF-R protein or proteincompositions, means that the protein or protein composition issubstantially free of other proteins of natural or endogenous origin andcontains less than about 1% by mass of protein contaminants residual ofproduction processes. Such compositions, however, can contain otherproteins added as stabilizers, carriers, excipients or co-therapeutics.TNF-R is isolated if it is detectable as a single protein band in apolyacrylamide gel by silver staining.

The term “substantially similar,” when used to define either amino acidor nucleic acid sequences, means that a particular subject sequence, forexample, a mutant sequence, varies from a reference sequence by one ormore substitutions, deletions, or additions, the net effect of which isto retain biological activity of the TNF-R protein as may be determined,for example, in one of the TNF-R binding assays set forth in Example 1below. Alternatively, nucleic acid subunits and analogs are“substantially similar” to the specific DNA sequences disclosed hereinif: (a) the DNA sequence is derived from the coding region of a nativemammalian TNF-R gene; (b) the DNA sequence is capable of hybridizationto DNA sequences of (a) under moderately stringent conditions (50° C.,2×SSC) and which encode biologically active TNF-R molecules; or DNAsequences which are degenerate as a result of the genetic code to theDNA sequences defined in (a) or (b) and which encode biologically activeTNF-R molecules.

“Recombinant,” as used herein, means that a protein is derived fromrecombinant (e.g., microbial or mammalian) expression systems.“Microbial” refers to recombinant proteins made in bacterial or fungal(e.g., yeast) expression systems. As a product, “recombinant microbial”defines a protein produced in a microbial expression system which isessentially free of native endogenous substances. Protein expressed inmost bacterial cultures, e.g., E. coli, will be free of glycan. Proteinexpressed in yeast may have a glycosylation pattern different from thatexpressed in mammalian cells.

“Biologically active,” as used throughout the specification as acharacteristic of TNF receptors, means that a particular molecule sharessufficient amino acid sequence similarity with the embodiments of thepresent invention disclosed herein to be capable of binding detectablequantities of TNF, transmitting a TNF stimulus to a cell, for example,as a component of a hybrid receptor construct, or cross-reacting withanti-TNF-R antibodies raised against TNF-R from natural (i.e.,nonrecombinant) sources. Preferably, biologically active TNF receptorswithin the scope of the present invention are capable of binding greaterthan 0.1 nmoles TNF per nmole receptor, and most preferably, greaterthan 0.5 nmole TNF per nmole receptor in standard binding assays (seebelow).

“Isolated DNA sequence” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesequence and its component nucleotide sequences by standard biochemicalmethods, for example, using a cloning vector. Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences, or introns, which are typicallypresent in eukaryotic genes. Genomic DNA containing the relevantsequences could also be used as a source of coding sequences. Sequencesof non-translated DNA may be present 5′ or 3′ from the open readingframe, where the same do not interfere with manipulation or expressionof the coding regions.

“Nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides.DNA sequences encoding the proteins provided by this invention can beassembled from cDNA fragments and short oligonucleotide linkers, or froma series of oligonucleotides, to provide a synthetic gene which iscapable of being expressed in a recombinant transcriptional unit.

Isolation of cDNAs Encoding TNF-R

The coding sequence of TNF-R is obtained by isolating a complementaryDNA (cDNA) sequence encoding TNF-R from a recombinant cDNA or genomicDNA library. A cDNA library is preferably constructed by obtainingpolyadenylated mRNA from a particular cell line which expresses amammalian TNF-R, for example, the human fibroblast cell line WI-26 VA4(ATCC CCL 95.1) and using the mRNA as a template for synthesizing doublestranded cDNA. The double stranded cDNA is then packaged into arecombinant vector, which is introduced into an appropriate E. colistrain and propagated. Murine or other mammalian cell lines whichexpress TNF-R may also be used. TNF-R sequences contained in the cDNAlibrary can be readily identified by screening the library with anappropriate nucleic acid probe which is capable of hybridizing withTNF-R cDNA. Alternatively, DNAs encoding TNF-R proteins can be assembledby ligation of synthetic oligonucleotide subunits corresponding to allor part of the sequence of SEQ ID NO:1 or SEQ ID NO:3 to provide acomplete coding sequence.

The human TNF receptor cDNAs of the present invention were isolated bythe method of direct expression cloning. A cDNA library was constructedby first isolating cytoplasmic mRNA from the human fibroblast cell lineWI-26 VA4. Polyadenylated RNA was isolated and used to preparedouble-stranded cDNA. Purified cDNA fragments were then ligated intopCAV/NOT vector DNA which uses regulatory sequences derived from pDC201(a derivative of pMLSV, previously described by Cosman et al., Nature312: 768, 1984), SV40 and cytomegalovirus DNA, described in detail belowin Example 2. pCAV/NOT has been deposited with the American Type CultureCollection under accession No. ATCC 68014. The pCAV/NOT vectorscontaining the W126-VA4 cDNA fragments were transformed into E. colistrain DH5α. Transformants were plated to provide approximately 800colonies per plate. The resulting colonies were harvested and each poolused to prepare plasmid DNA for transfection into COS-7 cellsessentially as described by Cosman et al. (Nature 312: 768, 1984) andLuthman et al. (Nucl. Acid Res. 11: 1295, 1983). Transformantsexpressing biologically active cell surface TNF receptors wereidentified by screening for their ability to bind ¹²⁵I-TNF. In thisscreening approach, transfected COS-7 cells were incubated with mediumcontaining ¹²⁵I-TNF, the cells washed to remove unbound labeled TNF, andthe cell monolayers contacted with X-ray film to detect concentrationsof TNF binding, as disclosed by Sims et al, Science 241: 585 (1988).Transfectants detected in this manner appear as dark foci against arelatively light background.

Using this approach, approximately 240,000 cDNAs were screened in poolsof approximately 800 cDNAs until assay of one transfectant poolindicated positive foci for TNF binding. A frozen stock of bacteria fromthis positive pool was grown in culture and plated to provide individualcolonies, which were screened until a single clone (clone 1) wasidentified which was capable of directing synthesis of a surface proteinwith detectable TNF binding activity. The sequence of cDNA clone 1isolated by the above method is depicted in SEQ ID NO:1.

Additional cDNA clones can be isolated from cDNA libraries of othermammalian species by cross-species hybridization. For use inhybridization, DNA encoding TNF-R may be covalently labeled with adetectable substance such as a fluorescent group, a radioactive atom ora chemiluminescent group by methods well known to those skilled in theart. Such probes could also be used for in vitro diagnosis of particularconditions.

Like most mammalian genes, mammalian TNF receptors are presumablyencoded by multi-exon genes. Alternative mRNA constructs which can beattributed to different mRNA splicing events following transcription,and which share large regions of identity or similarity with the cDNAsclaimed herein, are considered to be within the scope of the presentinvention.

Other mammalian TNF-R cDNAs are isolated by using an appropriate humanTNF-R DNA sequence as a probe for screening a particular mammalian cDNAlibrary by cross-species hybridization.

Proteins and Analogs

The present invention provides isolated recombinant mammalian TNF-Rpolypeptides. Isolated TNF-R polypeptides of this invention aresubstantially free of other contaminating materials of natural orendogenous origin and contain less than about 1% by mass of proteincontaminants residual of production processes. The native human TNF-Rmolecules are recovered from cell lysates as glycoproteins having anapparent molecular weight by SDS-PAGE of about 80 kilodaltons (kDa). TheTNF-R polypeptides of this invention are optionally without associatednative-pattern glycosylation.

Mammalian TNF-R of the present invention includes, by way of example,primate, human, murine, canine, feline, bovine, ovine, equine andporcine TNF-R. Mammalian TNF-Rs can be obtained by cross specieshybridization, using a single stranded cDNA derived from the human TNF-RDNA sequence as a hybridization probe to isolate TNF-R cDNAs frommammalian cDNA libraries.

Derivatives of TNF-R within the scope of the invention also includevarious structural forms of the primary protein which retain biologicalactivity. Due to the presence of ionizable amino and carboxyl groups,for example, a TNF-R protein may be in the form of acidic or basicsalts, or may be in neutral form. Individual amino acid residues mayalso be modified by oxidation or reduction.

The primary amino acid structure may be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like, or by creatingamino acid sequence mutants. Covalent derivatives are prepared bylinking particular functional groups to TNF-R amino acid side chains orat the N- or C-termini. Other derivatives of TNF-R within the scope ofthis invention include covalent or aggregative conjugates of TNF-R orits fragments with other proteins or polypeptides, such as by synthesisin recombinant culture as N-terminal or C-terminal fusions. For example,the conjugated peptide may be a signal (or leader) polypeptide sequenceat the N-terminal region of the protein which co-translationally orpost-translationally directs transfer of the protein from its site ofsynthesis to its site of function inside or outside of the cell membraneor wall (e.g., the yeast α-factor leader). TNF-R protein fusions cancomprise peptides added to facilitate purification or identification ofTNF-R (e.g., poly-His). The amino acid sequence of TNF receptor can alsobe linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK (SEQID NO:22)) (Hopp et al., Bio/Technology 6: 1204, 1988.) The lattersequence is highly antigenic and provides an epitope reversibly bound bya specific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. This sequence is alsospecifically cleaved by bovine mucosal enterokinase at the residueimmediately following the Asp-Lys pairing. Fusion proteins capped withthis peptide may also be resistant to intracellular degradation in E.coli.

TNF-R derivatives may also be used as immunogens, reagents inreceptor-based immunoassays, or as binding agents for affinitypurification procedures of TNF or other binding ligands. TNF-Rderivatives may also be obtained by cross-linking agents, such asM-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, atcysteine and lysine residues. TNF-R proteins may also be covalentlybound through reactive side groups to various insoluble substrates, suchas cyanogen bromide-activated, bisoxirane-activated,carbonyldiimidazole-activated or tosyl-activated agarose structures, orby adsorbing to polyolefin surfaces (with or without glutaraldehydecross-linking). Once bound to a substrate, TNF-R may be used toselectively bind (for purposes of assay or purification) anti-TNF-Rantibodies or TNF.

The present invention also includes TNF-R with or without associatednative-pattern glycosylation. TNF-R expressed in yeast or mammalianexpression systems, e.g., COS-7 cells, may be similar or slightlydifferent in molecular weight and glycosylation pattern than the nativemolecules, depending upon the expression system. Expression of TNF-RDNAs in bacteria such as E. coli provides non-glycosylated molecules.Functional mutant analogs of mammalian TNF-R having inactivatedN-glycosylation sites can be produced by oligonucleotide synthesis andligation or by site-specific mutagenesis techniques. These analogproteins can be produced in a homogeneous, reduced-carbohydrate form ingood yield using yeast expression systems. N-glycosylation sites ineukaryotic proteins are characterized by the amino acid tripletAsn-A₁-Z, where A₁ is any amino acid except Pro, and Z is Ser or Thr. Inthis sequence, asparagine provides a side chain amino group for covalentattachment of carbohydrate. Such a site can be eliminated bysubstituting another amino acid for Asn or for residue Z, deleting Asnor Z, or inserting a non-Z amino acid between A₁ and Z, or an amino acidother than Asn between Asn and A₁.

TNF-R derivatives may also be obtained by mutations of TNF-R or itssubunits. A TNF-R mutant, as referred to herein, is a polypeptidehomologous to TNF-R but which has an amino acid sequence different fromnative TNF-R because of a deletion, insertion or substitution.

Bioequivalent analogs of TNF-R proteins may be constructed by, forexample, making various substitutions of residues or sequences ordeleting terminal or internal residues or sequences not needed forbiological activity. For example, cysteine residues can be deleted(e.g., Cys¹⁷⁸) or replaced with other amino acids to prevent formationof unnecessary or incorrect intramolecular disulfide bridges uponrenaturation. Other approaches to mutagenesis involve modification ofadjacent dibasic amino acid residues to enhance expression in yeastsystems in which KEX2 proteasc activity is present. Generally,substitutions should be made conservatively; i.e., the most preferredsubstitute amino acids are those having physiochemical characteristicsresembling those of the residue to be replaced. Similarly, when adeletion or insertion strategy is adopted, the potential effect of thedeletion or insertion on biological activity should be considered.Substantially similar polypeptide sequences, as defined above, generallycomprise a like number of amino acids sequences, although C-terminaltruncations for the purpose of constructing soluble TNF-Rs will containfewer amino acid sequences. In order to preserve the biological activityof TNF-Rs, deletions and substitutions will preferably result inhomologous or conservatively substituted sequences, meaning that a givenresidue is replaced by a biologically similar residue. Examples ofconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another, orsubstitutions of one polar residue for another, such as between Lys andArg; Glu and Asp; or Gln and Asn. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity characteristics, are well known. Moreover, particularamino acid differences between human, murine and other mammalian TNF-Rsis suggestive of additional conservative substitutions that may be madewithout altering the essential biological characteristics of TNF-R.

Subunits of TNF-R may be constructed by deleting terminal or internalresidues or sequences. Particularly preferred sequences include those inwhich the transmembrane region and intracellular domain of TNF-R aredeleted or substituted with hydrophilic residues to facilitate secretionof the receptor into the cell culture medium. The resulting protein isreferred to as a soluble TNF-R molecule which retains its ability tobind TNF. A particularly preferred soluble TNF-R construct is TNF-RΔ235(the sequence of amino acids 1-235 of SEQ ID NO:1), which comprises theentire extracellular region of TNF-R, terminating with Asp²³⁵immediately adjacent the transmembrane region. Additional amino acidsmay be deleted from the extracellular region while retaining TNF bindingactivity. For example, huTNF-RΔ183 which comprises the sequence of aminoacids 1-183 of SEQ ID NO:1, and TNF-RΔ163 which comprises the sequenceof amino acids 1-163 of SEQ ID NO:1, retain the ability to bind TNFligand as determined using the binding assays described below inExample 1. TNF-RΔ142, however, does not retain the ability to bind TNFligand. This suggests that one or both of Cys¹⁵⁷ and Cys¹⁶³ is requiredfor formation of an intramolecular disulfide bridge for the properfolding of TNF-R. Cys¹⁷⁸, which was deleted without any apparent adverseeffect on the ability of the soluble TNF-R to bind TNF, does not appearto be essential for proper folding of TNF-R. Thus, any deletionC-terminal to Cys¹⁶³ would be expected to result in a biologicallyactive soluble TNF-R. The present invention contemplates such solubleTNF-R constructs corresponding to all or part of the extracellularregion of TNF-R terminating with any amino acid after Cys¹⁶³. OtherC-terminal deletions, such as TNF-FΔ157, may be made as a matter ofconvenience by cutting TNF-R cDNA with appropriate restriction enzymesand, if necessary, reconstructing specific sequences with syntheticoligonucleotide linkers. The resulting soluble TNF-R constructs are theninserted and expressed in appropriate expression vectors and assayed forthe ability to bind TNF, as described in Example 1. Biologically activesoluble TNF-Rs resulting from such constructions are also contemplatedto be within the scope of the present invention.

Mutations in nucleotide sequences constructed for expression of analogTNF-R must, of course, preserve the reading frame phase of the codingsequences and preferably will not create complementary regions thatcould hybridize to produce secondary mRNA structures such as loops orhairpins which would adversely affect translation of the receptor mRNA.Although a mutation site may be predetermined, it is not necessary thatthe nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed TNF-R mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes TNF-R will beexpressed in the final product, for example, nucleotide substitutionsmay be made to enhance expression, primarily to avoid secondarystructure loops in the transcribed mRNA (see EPA 75,444A, incorporatedherein by reference), or to provide codons that are more readilytranslated by the selected host, e.g., the well-known E. coli preferencecodons for E. coli expression.

Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene37: 73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

Both monovalent forms and polyvalent forms of TNF-R are useful in thecompositions and methods of this invention. Polyvalent forms possessmultiple TNF-R binding sites for TNF ligand. For example, a bivalentsoluble TNF-R may consist of two tandem repeats of amino acids 1-235 ofSEQ ID NO: 1, separated by a linker region. Alternate polyvalent formsmay also be constructed, for example, by chemically coupling TNF-R toany clinically acceptable carrier molecule, a polymer selected from thegroup consisting of Ficoll, polyethylene glycol or dextran usingconventional coupling techniques. Alternatively, TNF-R may be chemicallycoupled to biotin, and the biotin-TNF-R conjugate then allowed to bindto avidin, resulting in tetravalent avidin/biotin/TNF-R molecules. TNF-Rmay also be covalently coupled to dinitrophenol (DNP) or trinitrophenol(TNP) and the resulting conjugate precipitated with anti-DNP oranti-TNP-IgM, to form decameric conjugates with a valency of 10 forTNF-R binding sites.

A recombinant chimeric antibody molecule may also be produced havingTNF-R sequences substituted for the variable domains of either or bothof the immunoglobulin molecule heavy and light chains and havingunmodified constant region domains. For example, chimeric TNF-R/IgG₁ maybe produced from two chimeric genes—a TNF-R/human κ light chain chimera(TNF-R/C_(κ)) and a TNF-R/human γ₁ heavy chain chimera (TNF-R/C_(γ-1)).Following transcription and translation of the two chimeric genes, thegene products assemble into a single chimeric antibody molecule havingTNF-R displayed bivalently. Such polyvalent forms of TNF-R may haveenhanced binding affinity for TNF ligand. Additional details relating tothe construction of such chimeric antibody molecules are disclosed in WO89/09622 and EP 315062.

Expression of Recombinant TNF-R

The present invention provides recombinant expression vectors to amplifyor express DNA encoding TNF-R. Recombinant expression vectors arereplicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding mammalian TNF-R or bioequivalent analogs operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, microbial, viral or insect genes. Atranscriptional unit generally comprises an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements may include an operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants mayadditionally be incorporated. DNA regions are operably linked when theyare functionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operably linked to DNA for a polypeptideif it is expressed as a precursor which participates in the secretion ofthe polypeptide; a promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; or a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto permit translation. Generally, operably linked means contiguous and,in the case of secretory leaders, contiguous and in reading frame.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

DNA sequences encoding mammalian TNF receptors which are to be expressedin a microorganism will preferably contain no introns that couldprematurely terminate transcription of DNA into mRNA; however, prematuretermination of transcription may be desirable, for example, where itwould result in mutants having advantageous C-terminal truncations, forexample, deletion of a transmembrane region to yield a soluble receptornot bound to the cell membrane. Due to code degeneracy, there can beconsiderable variation in nucleotide sequences encoding the same aminoacid sequence. Other embodiments include sequences capable ofhybridizing to the sequences of the provided cDNA under moderatelystringent conditions (50° C., 2×SSC) and other sequences hybridizing ordegenerate to those which encode biologically active TNF receptorpolypeptides.

Recombinant TNF-R DNA is expressed or amplified in a recombinantexpression system comprising a substantially homogeneous monoculture ofsuitable host microorganisms, for example, bacteria such as E. coli oryeast such as S. cerevisiae, which have stably integrated (bytransformation or transfection) a recombinant transcriptional unit intochromosomal DNA or carry the recombinant transcriptional unit as acomponent of a resident plasmid. Generally, cells constituting thesystem are the progeny of a single ancestral transformant. Recombinantexpression systems as defined herein will express heterologous proteinupon induction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

Transformed host cells are cells which have been transformed ortransfected with TNF-R vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express TNF-R, but hostcells transformed for purposes of cloning or amplifying TNF-R DNA do notneed to express TNF-R. Expressed TNF-R will be deposited in the cellmembrane or secreted into the culture supernatant, depending on theTNF-R DNA selected. Suitable host cells for expression of mammalianTNF-R include prokaryotes, yeast or higher eukaryotic cells under thecontrol of appropriate promoters. Prokaryotes include gram negative orgram positive organisms, for example E. coli or bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed toproduce mammalian TNF-R using RNAs derived from the DNA constructs ofthe present invention. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwels et al. (Cloning Vectors: A Laboratory Manual,Elsevier, New York, 1985), the relevant disclosure of which is herebyincorporated by reference.

Prokaryotic expression hosts may be used for expression of TNF-R that donot require extensive proteolytic and disulfide processing. Prokaryoticexpression vectors generally comprise one or more phenotypic selectablemarkers, for example a gene encoding proteins conferring antibioticresistance or supplying an autotrophic requirement, and an origin ofreplication recognized by the host to ensure amplification within thehost. Suitable prokaryotic hosts for transformation include E. coli,Bacillus subtilis, Salmonella typhimurium, and various species withinthe genera Pseudomonas, Streptomyces, and Staphylococcus, althoughothers may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. E. coli is typically transformed using derivatives ofpBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene2: 95, 1977). pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275: 615, 1978; and Goeddel et al., Nature 281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8: 4057, 1980; and EPA 36,776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful bacterial expression system employsthe phage λ P_(L) promoter and cI857ts thermolabile repressor. Plasmidvectors available from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

Recombinant TNF-R proteins may also be expressed in yeast hosts,preferably from the Saccharomyces species, such as S. cerevisiae. Yeastof other genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS),promoter, DNA encoding TNF-R, sequences for polyadenylation andtranscription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerpermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 or URA3gene, which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, and a promoter derived from ahighly expressed yeast gene to induce transcription of a structuralsequence downstream. The presence of the TRP1 or URA3 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan oruracil.

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255: 2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7: 149, 1968; and Holland et al., Biochem. 17: 4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pUC18for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beieret al. (Nature 300: 724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30: 933, 1982; and Bitter et al., Proc. Natl. Acad Sci. USA 81: 5330,1984. The leader sequence may be modified to contain, near its 3′ end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

Suitable yeast transformation protocols are known to those of skill inthe art; an exemplary technique is described by Hinnen et al., Proc.Natl. Acad Sci. USA 75: 1929, 1978, selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil or URA+tranformants in medium consisting of 0.67% YNB, with amino acids andbases as described by Sherman et al., Laboratory Course Manual forMethods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1986.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% or 4% glucose supplemented with 80 μg/ml adenine and 80μg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustionof medium glucose. Crude yeast supernatants are harvested by filtrationand held at 4° C. prior to further purification.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells is particularly preferred because suchproteins are generally correctly folded, appropriately modified andcompletely functional. Examples of suitable mammalian host cell linesinclude the COS-7 lines of monkey kidney cells, described by Gluzman(Cell 23: 175, 1981), and other cell lines capable of expressing anappropriate vector including, for example, L cells, C127, 3T3, Chinesehamster ovary (CHO), HeLa and BHK cell lines. Mammalian expressionvectors may comprise nontranscribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, and other 5′ or 3′ flanking nontranscribed sequences, and 5′or 3′ nontranslated sequences, such as necessary ribosome binding sites,a polyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6: 47 (1988).

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., Nature 273: 113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind 3 site toward the Bgl1 site located inthe viral origin of replication is included. Further, mammalian genomicTNF-R promoter, control and/or signal sequences may be utilized,provided such control sequences are compatible with the host cellchosen. Additional details regarding the use of a mammalian highexpression vector to produce a recombinant mammalian TNF receptor areprovided in Examples 2 and 7 below. Exemplary vectors can be constructedas disclosed by Okayama and Berg (Mol. Cell. Biol. 3: 280,1983).

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23: 935,1986).

In preferred aspects of the present invention, recombinant expressionvectors comprising TNF-R cDNAs are stably integrated into a host cell'sDNA. Elevated levels of expression product is achieved by selecting forcell lines having amplified numbers of vector DNA. Cell lines havingamplified numbers of vector DNA are selected, for example, bytransforming a host cell with a vector comprising a DNA sequence whichencodes an enzyme which is inhibited by a known drug. The vector mayalso comprise a DNA sequence which encodes a desired protein.Alternatively, the host cell may be co-transformed with a second vectorwhich comprises the DNA sequence which encodes the desired protein. Thetransformed or co-transformed host cells are then cultured in increasingconcentrations of the known drug, thereby selecting for drug-resistantcells. Such drug-resistant cells survive in increased concentrations ofthe toxic drug by over-production of the enzyme which is inhibited bythe drug, frequently as a result of amplification of the gene encodingthe enzyme. Where drug resistance is caused by an increase in the copynumber of the vector DNA encoding the inhibitable enzyme, there is aconcomitant co-amplification of the vector DNA encoding the desiredprotein (TNF-R) in the host cell's DNA.

A preferred system for such co-amplification uses the gene fordihydrofolate reductase (DHFR), which can be inhibited by the drugmethotrexate (MTX). To achieve co-amplification, a host cell which lacksan active gene encoding DHFR is either transformed with a vector whichcomprises DNA sequence encoding DHFR and a desired protein, or isco-transformed with a vector comprising a DNA sequence encoding DHFR anda vector comprising a DNA sequence encoding the desired protein. Thetransformed or co-transformed host cells are cultured in mediacontaining increasing levels of MTX, and those cells lines which surviveare selected.

A particularly preferred co-amplification system uses the gene forglutamine synthetase (GS), which is responsible for the synthesis ofglutamate and ammonia using the hydrolysis of ATP to ADP and phosphateto drive the reaction. GS is subject to inhibition by a variety ofinhibitors, for example methionine sulphoximine (MSX). Thus, TNF-R canbe expressed in high concentrations by co-amplifying cells transformedwith a vector comprising the DNA sequence for GS and a desired protein,or co-transformed with a vector comprising a DNA sequence encoding GSand a vector comprising a DNA sequence encoding the desired protein,culturing the host cells in media containing increasing levels of MSXand selecting for surviving cells. The GS co-amplification system,appropriate recombinant expression vectors and cells lines, aredescribed in the following PCT applications: WO 87/04462, WO 89/01036,WO 89/10404 and WO 86/05807.

Recombinant proteins are preferably expressed by co-amplification ofDHFR or GS in a mammalian host cell, such as Chinese Hamster Ovary (CHO)cells, or alternatively in a murine myeloma cell line, such asSP2/0-Ag14 or NS0 or a rat myeloma cell line, such as YB2/3.0-Ag20,disclosed in PCT applications WO/89/10404 and WO 86/05807.

A preferred eukaryotic vector for expression of TNF-R DNA is disclosedbelow in Example 2. This vector, referred to as pCAV/NOT, was derivedfrom the mammalian high expression vector pDC201 and contains regulatorysequences from SV40, adenovirus-2, and human cytomegalovirus.

Purification of Recombinant TNF-R

Purified mammalian TNF receptors or analogs are prepared by culturingsuitable host/vector systems to express the recombinant translationproducts of the DNAs of the present invention, which are then purifiedfrom culture media or cell extracts.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.For example, a suitable affinity matrix can comprise a TNF or lectin orantibody molecule bound to a suitable support. Alternatively, an anionexchange resin can be employed, for example, a matrix or substratehaving pendant diethylaminoethyl (DEAE) groups. The matrices can beacrylamide, agarose, dextran, cellulose or other types commonly employedin protein purification. Alternatively, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups arepreferred.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a TNF-R composition. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant mammalian TNF-R can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express mammalian TNF-R as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296: 171,1984). This reference describes two sequential, reversed-phase HPLCsteps for purification of recombinant human GM-CSF on a preparative HPLCcolumn.

Human TNF-R synthesized in recombinant culture is characterized by thepresence of non-human cell components, including proteins, in amountsand of a character which depend upon the purification steps taken torecover human TNF-R from the culture. These components ordinarily willbe of yeast, prokaryotic or non-human higher eukaryotic origin andpreferably are present in innocuous contaminant quantities, on the orderof less than about 1 percent by weight. Further, recombinant cellculture enables the production of TNF-R free of proteins which may benormally associated with TNF-R as it is found in nature in its speciesof origin, e.g. in cells, cell exudates or body fluids.

Therapeutic Administration of Recombinant Soluble TNF-R

The present invention provides methods of using therapeutic compositionscomprising an effective amount of soluble TNF-R proteins and a suitablediluent and carrier, and methods for suppressing TNF-dependentinflammatory responses in humans comprising administering an effectiveamount of soluble TNF-R protein.

For therapeutic use, purified soluble TNF-R protein is administered to apatient, preferably a human, for treatment in a manner appropriate tothe indication. Thus, for example, soluble TNF-R protein compositionscan be administered by bolus injection, continuous infusion, sustainedrelease from implants, or other suitable technique. Typically, a solubleTNF-R therapeutic agent will be administered in the form of acomposition comprising purified protein in conjunction withphysiologically acceptable carriers, excipients or diluents. Suchcarriers will be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the TNF-R with buffers, antioxidants suchas ascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose or dextrins, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.Preferably, product is formulated as a lyophilizate using appropriateexcipient solutions (e.g., sucrose) as diluents. Appropriate dosages canbe determined in trials. The amount and frequency of administration willdepend, of course, on such factors as the nature and severity of theindication being treated, the desired response, the condition of thepatient, and so forth.

Soluble TNF-R proteins are administered for the purpose of inhibitingTNF-dependent responses. A variety of diseases or conditions arebelieved to be caused by TNF, such as cachexia and septic shock. Inaddition, other key cytokines (IL-1, IL-2 and other colony stimulatingfactors) can also induce significant host production of TNF. SolubleTNF-R compositions may therefore be used, for example, to treat cachexiaor septic shock or to treat side effects associated with cytokinetherapy. Because of the primary roles IL-1 and IL-2 play in theproduction of TNF, combination therapy using both IL-1 receptors or IL-2receptors may be preferred in the treatment of TNF-associated clinicalindications.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Binding Assays

A. Radiolabeling of TNFα and TNFβ. Recombinant human TNFα, in the formof a fusion protein containing a hydrophilic octapeptide at theN-terminus, was expressed in yeast as a secreted protein and purified byaffinity chromatography (Hopp et al., Bio/Technology 6: 1204, 1988).Purified recombinant human TNFβ was purchased from R&D Systems(Minneapolis, Minn.). Both proteins were radiolabeled using thecommercially available solid phase agent, IODO-GEN (Pierce). In thisprocedure, 5 μg of IODO-GEN were plated at the bottom of a 10×75 mmglass tube and incubated for 20 minutes at 4° C. with 75 μl of 0.1 Msodium phosphate, pH 7.4 and 20 μl (2 mCi) Na ¹²⁵I. This solution wasthen transferred to a second glass tube containing 5 μg TNFα (or TNFβ)in 45 μl PBS for 20 minutes at 4° C. The reaction mixture wasfractionated by gel filtration on a 2 ml bed volume of Sephadex G-25(Sigma) equilibrated in Roswell Park Memorial Institute (RPMI) 1640medium containing 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v)sodium azide and 20 mM Hepes pH 7.4 (binding medium). The final pool of¹²⁵I-TNF was diluted to a working stock solution of 1×10⁻⁷ M in bindingmedium and stored for up to one month at 4° C. without detectable lossof receptor binding activity. The specific activity is routinely 1×10⁶cpm/mmole TNF.

B. Binding to Intact Cells. Binding assays with intact cells wereperformed by two methods. In the first method, cells were first growneither in suspension (e.g., U 937) or by adherence on tissue cultureplates (e.g., W126-VA4, COS cells expressing the recombinant TNFreceptor). Adherent cells were subsequently removed by treatment with 5mM EDTA treatment for ten minutes at 37 degrees centigrade. Bindingassays were then performed by a phthalate oil separation method (Doweret al., J. Immunol. 132: 751, 1984) essentially as described by Park etal. (J. Biol. Chem. 261: 4177, 1986). Non-specific binding of ¹²⁵I-TNFwas measured in the presence of a 200-fold or greater molar excess ofunlabeled TNF. Sodium azide (0.2%) was included in a binding assay toinhibit internalization of ¹²⁵I-TNF by cells. In the second method, COScells transfected with the TNF-R-containing plasmid, and expressing TNFreceptors on the surface, were tested for the ability to bind ¹²⁵I-TNFby the plate binding assay described by Sims et al. (Science 241: 585,1988).

C. Solid Phase Binding Assays. The ability of TNF-R to be stablyadsorbed to nitrocellulose from detergent extracts of human cells yetretain TNF-binding activity provided a means of detecting TNF-R. Cellextracts were prepared by mixing a cell pellet with a 2×volume of PBScontaining 1% Triton X-100 and a cocktail of protease inhibitors (2 mMphenylmethyl sulfonyl fluoride, 10 μM pepstatin, 10 μM leupeptin, 2 mMo-phenanthroline and 2 mM EGTA) by vigorous vortexing. The mixture wasincubated on ice for 30 minutes after which it was centrifuged at12,000×g for 15 minutes at 8° C. to remove nuclei and other debris. Twomicroliter aliquots of cell extracts were placed on dry BA85/21nitrocellulose membranes (Schleicher and Schuell, Keene, N H) andallowed to dry. The membranes were incubated in tissue culture dishesfor 30 minutes in Tris (0.05 M) buffered saline (0.15 M) pH 7.5containing 3% w/v BSA to block nonspecific binding sites. The membranewas then covered with 5×10⁻¹¹ M ¹²⁵I-TNF in PBS+3% BSA and incubated for2 hr at 4° C. with shaking. At the end of this time, the membranes werewashed 3 times in PBS, dried and placed on Kodak X-Omat AR film for 18hr at −70° C.

Example 2 Isolation of Human TNF-R cDNA by Direct Expression of ActiveProtein in COS-7 Cells

Various human cell lines were screened for expression of TNF-R based ontheir ability to bind ¹²⁵I-labeled TNF. The human fibroblast cell lineWI-26 VA4 was found to express a reasonable number of receptors percell. Equilibrium binding studies showed that the cell line exhibitedbiphasic binding of ¹²⁵I-TNF with approximately 4,000 high affinitysites (K_(a)=1×10¹⁰ M⁻¹) and 15,00 low affinity sites (K_(a)=1×10⁸ M⁻¹)per cell.

An unsized cDNA library was constructed by reverse transcription ofpolyadenylated mRNA isolated from total RNA extracted from humanfibroblast WI-26 VA4 cells grown in the presence of pokeweed mitogenusing standard techniques (Gubler, et al., Gene 25: 263, 1983; Ausubelet al., eds., Current Protocols in Molecular Biology, Vol. 1, 1987). Thecells were harvested by lysing the cells in a guanidine hydrochloridesolution and total RNA isolated as previously described (March et al.,Nature 315: 641, 1985).

Poly A⁺ RNA was isolated by oligo dT cellulose chromatography anddouble-stranded cDNA was prepared by a method similar to that of Gublerand Hoffman (Gene 25: 263, 1983). Briefly, the poly A⁺ RNA was convertedto an RNA-cDNA hybrid by reverse transcriptase using oligo dT as aprimer. The RNA-cDNA hybrid was then converted into double-stranded cDNAusing RNAase H in combination with DNA polymerase I. The resultingdouble stranded cDNA was blunt-ended with T4 DNA polymerase. To theblunt-ended cDNA is added EcoRI linker-adapters (having internal Not1sites) which were phosphorylated on only one end (Invitrogen). Thelinker-adaptered cDNA was treated with T4 polynucleotide kinase tophosphorylate the 5′ overhanging region of the linker-adapter andunligated linkers were removed by running the cDNA over a Sepharose CL4Bcolumn. The linker-adaptered cDNA was ligated to an equimolarconcentration of EcoR1 cut and dephosphorylated arms of bacteriophageλkgt10 (Huynh et al, DNA Cloning: A Practical Approach, Glover, ed., IRLPress, pp. 49-78). The ligated DNA was packaged into phage particlesusing a commercially available kit to generate a library of recombinants(Stratagene Cloning Systems, San Diego, Calif., USA). Recombinants werefurther amplified by plating phage on a bacterial lawn of E. coli strainc600(hfl⁻).

Phage DNA was purified from the resulting λgt10 cDNA library and thecDNA inserts excised by digestion with the restriction enzyme Not1.Following electrophoresis of the digest through an agarose gel, cDNAsgreater than 2,000 bp were isolated.

The resulting cDNAs were ligated into the eukaryotic expression vectorpCAV/NOT, which was designed to express cDNA sequences inserted at itsmultiple cloning site when transfected into mammalian cells. pCAV/NOTwas assembled from pDC201 (a derivative of pMLSV, previously describedby Cosman et al., Nature 312: 768, 1984), SV40 and cytomegalovirus DNAand comprises, in sequence with the direction of transcription from theorigin of replication: (1) SV40 sequences from coordinates 5171-270including the origin of replication, enhancer sequences and early andlate promoters; (2) cytomegalovirus sequences including the promoter andenhancer regions (nucleotides 671 to +63 from the sequence published byBoechart et al. (Cell 41: 521, 1985); (3) adenovirus-2 sequencescontaining the first exon and part of the intron between the first andsecond exons of the tripartite leader, the second exon and part of thethird exon of the tripartite leader and a multiple cloning site (MCS)containing sites for Xho1, Kpn1, Sma1, Not1 and Bgl1; (4) SV40 sequencesfrom coordinates 4127-4100 and 2770-2533 that include thepolyadenylation and termination signals for early transcription; (5)sequences derived from pBR322 and virus-associated sequences VAI andVAII of pDC201, with adenovirus sequences 10532-11156 containing the VAIand VAII genes, followed by pBR322 sequences from 4363-2486 and 1094-375containing the ampicillin resistance gene and origin of replication.

The resulting WI-26 VA4 cDNA library in pCAV/NOT was used to transformE. coli strain DH5α, and recombinants were plated to provideapproximately 800 colonies per plate and sufficient plates to provideapproximately 50,000 total colonies per screen. Colonies were scrapedfrom each plate, pooled, and plasmid DNA prepared from each pool. Thepooled DNA was then used to transfect a sub-confluent layer of monkeyCOS-7 cells using DEAE-dextran followed by chloroquine treatment, asdescribed by Luthman et al. (Nucl. Acids Res. 11: 1295, 1983) andMcCutchan et al. (J. Natl. Cancer Inst. 41: 351, 1986). The cells werethen grown in culture for three days to permit transient expression ofthe inserted sequences. After three days, cell culture supernatants werediscarded and the cell monolayers in each plate assayed for TNF bindingas follows. Three ml of binding medium containing 1.2×10⁻¹¹ M¹²⁵I-labeled FLAG®-TNF was added to each plate and the plates incubatedat 4° C. for 120 minutes. This medium was then discarded, and each platewas washed once with cold binding medium (containing no labeled TNF) andtwice with cold PBS. The edges of each plate were then broken off,leaving a flat disk which was contacted with X-ray film for 72 hours at−70° C. using an intensifying screen. TNF binding activity wasvisualized on the exposed films as a dark focus against a relativelyuniform background.

After approximately 240,000 recombinants from the library had beenscreened in this manner, one transfectant pool was observed to provideTNF binding foci which were clearly apparent against the backgroundexposure.

A frozen stock of bacteria from the positive pool was then used toobtain plates of approximately 150 colonies. Replicas of these plateswere made on nitrocellulose filters, and the plates were then scrapedand plasmid DNA prepared and transfected as described above to identifya positive plate. Bacteria from individual colonies from thenitrocellulose replica of this plate were grown in 0.2 ml cultures,which were used to obtain plasmid DNA, which was transfected into COS-7cells as described above. In this manner, a single clone, clone 1, wasisolated which was capable of inducing expression of human TNF-R in COScells. The expression vector pCAV/NOT containing the TNF-R cDNA clone 1has been deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, USA (Accession No. 68088) underthe name pCAV/NOT-TNF-R.

Example 3 Construction of cDNAs Encoding Soluble huTNF-RΔ235

A cDNA encoding a soluble huTNF-RΔ235 (having the sequence of aminoacids 1-235 of SEQ ID NO:1) was constructed by excising an 840 bpfragment from pCAV/NOT-TNF-R with the restriction enzymes Not1 and Pvu2.Not1 cuts at the multiple cloning site of pCAV/NOT-TNF-R and Pvu2 cutswithin the TNF-R coding region 20 nucleotides 5′ of the transmembraneregion. In order to reconstruct the 3′ end of the TNF-R sequences, twooligonucleotides (encoding amino acids corresponding to Ala²²⁹-Ala²³⁵ ofSEQ ID NO:1) were synthesized and annealed to create the followingoligonucleotide linker:

Pvu2                    BamH1 Bgl2 CTGAAGGGAGCACTGGCGACTAAGGATCCA (SEQID NO: 5 GACTTCCCTCGTGACCGCTGATTCCTAGGTCTAG (SEQ ID NO: 6)AlaGluGlySerThrGlyAspEnd (SEQ ID NO: 7)This oligonucleotide linker has terminal Pvu2 and Bgl2 restrictionsites, regenerates 20 nucleotides of the TNF-R, followed by atermination codon (underlined) and a BamH1 restriction site (forconvenience in isolating the entire soluble TNF-R by Not1/BamH1digestion). This oligonucleotide was then ligated with the 840 bpNot1/Pvu2 TNF-R insert into Bgl2/Notl cut pCAV/NOT to yieldpsolhuTNF-RΔ235/CAVNOT, which was transfected into COS-7 cells asdescribed above. This expression vector induced expression of solublehuman TNF-R which was capable of binding TNF.

Example 4 Construction of cDNAs Encoding Soluble huTNF-RΔ185

A cDNA encoding a soluble huTNF-RΔ185 (having the sequence of aminoacids 1-185 of SEQ ID NO:1) was constructed by excising a 640 bpfragment from pCAV/NOT-TNF-R with the restriction enzymes Not1 and Bgl2.Notl cuts at the multiple cloning site of pCAV/NO-TNF-R and Bgl2 cutswithin the TNF-R coding region at nucleotide 637, which is 237nucleotides 5′ of the transmembrane region. The followingoligonucleotide linkers (encoding amino acids corresponding toIle¹⁶²-Ala¹⁷⁶ and Val¹⁷⁷-Arg¹⁸⁵ of SEQ ID NO:1) were synthesized:

   Bg12 (SEQ ID NO: 8) 5′-GATCTGTAACGTGGTGGCCATCCCTGGGAATGCAAGCATGGATGC-3′ (SEQ ID NO: 9) ACATTGCACCACCGGTAGGGACCCTTACGTTCG (SEQ ID NO: 10)IleCysAsnValValAlaIleProGlyAsnAlaSerMetAspAla                                   Not 1 (SEQ ID NO: 11)5′-AGTCTGCACGTCCACGTCCCCCACCCGGTGAGC-3′ (SEQ ID NO: 12)TACCTACGTCAGACGTGCAGGTGCAGGGGGTGGGCCACTCGCCGG (SEQ ID NO: 13)ValCysThrSerThrSerProThrArgEndThe above oligonucleotide linkers reconstruct the 3′ end of the receptormolecule up to nucleotide 708, followed by a termination codon(underlined). These oligonucleotides were then ligated with the 640 bpNot1 TNF-R insert into Notl cut pCAV/NOT to yield the expression vectorpsolTNFRΔ185/CAVNOT, which was transfected into COS-7 cells as describedabove. This expression vector induced expression of soluble human TNF-Rwhich was capable of binding TNF.

Example 5 Construction of cDNAs Encoding Soluble huTNF-RA163

A cDNA encoding a soluble huTNF-RΔ163 (having the sequence of aminoacids 1-163 of SEQ ID NO:1) was constructed by excising a 640 bpfragment from from pCAV/NOT-TNF-R with the restriction enzymes Not1 andBgl2 as described in Example 4. The following oligonucleotide linkers(encoding amino acids corresponding to Ile¹⁶⁵-Cys¹⁶³ of SEQ ID NO:1)were synthesized:

   Bgl2     Not1 5′-GATCTGTTGAGC-3′ (SEQ ID NO: 14) ACAACTCGCCGG (SEQ IDNO: 15) IleCysEndThis above oligonucleotide linker reconstructs the 3′ end of thereceptor molecule up to nucleotide 642 (amino acid 163), followed by atermination codon (underlined). This oligonucleotide was then ligatedwith the 640 bp Not1 TNF-R insert into Notl cut pCAV/NOT to yield theexpression vector psolTNFRΔ163/CAVNOT, which was transfected into COS-7cells as described above. This expression vector induced expression ofsoluble human TNF-R which was capable of binding TNF in the bindingassay described in Example 1.

Example 6 Construction of cDNAs Encoding Soluble huTNF-RA142

A cDNA encoding a soluble huTNF-RΔ142 (having the sequence of aminoacids 1-142 of SEQ ID NO:1) was constructed by excising a 550 bpfragment from from pCAV/NOT-TNF-R with the restriction enzymes Not1 andAlwN1. AlwN1 cuts within the TNF-R coding region at nucleotide 549. Thefollowing oligonucleotide linker (encoding amino acids corresponding toThr¹³²-Cys¹⁴² of SEQ ID NO:1) was synthesized:

   Bgl2         Not1 (SEQ ID NO: 16)5′-CTGAAACATCAGACGTGGTGTGCAAGCCCTGTTAAA-3′ (SEQ ID NO: 17)CTTGACTTTGTAGTCTGCACCACACGTTCGGGACAATTTCTAGA                                   EndThis above oligonucleotide linker reconstructs the 3′ end of thereceptor molecule up to nucleotide 579 (amino acid 142), followed by atermination codon (underlined). This oligonucleotide was then ligatedwith the 550 bp Not1/AlwN1 TNF-R insert into Not1/Bgl2 cut pCAV/NOT toyield the expression vector psolTNFRΔ142/CAVNOT, which was transfectedinto COS-7 cells as described above. This expression vector did notinduce expression of soluble human TNF-R which was capable of bindingTNF. It is believed that this particular construct failed to expressbiologically active TNF-R because one or more essential cysteine residue(e.g., Cys¹⁵⁷ or Cys¹⁶³) required for intramolecular bonding (forformation of the proper tertiary structure of the TNF-R molecule) waseliminated.

Example 7 Expression of Soluble TNF Receptors in CHO Cells

Soluble TNF receptor was expressed in Chinese Hamster Ovary (CHO) cellsusing the glutamine-synthetase (GS) gene amplification system,substantially as described in PCT patent application Nos. WO87/04462 andWO89/01036. Briefly, CHO cells are transfected with an expression vectorcontaining genes for both TNF-R and GS. CHO cells are selected for GSgene expression based on the ability of the transfected DNA to conferresistance to low levels of methionine sulphoximine (MSX). GS sequenceamplification events in such cells are selected using elevated MSXconcentrations. In this way, contiguous TNF-R sequences are alsoamplified and enhanced TNF-R expression is achieved.

The vector used in the GS expression system was psolTNFR/P6/PSVLGS,which was constructed as follows. First, the vector pSVLGS.1 (describedin PCT Application Nos. WO87/04462 and WO89/01036, and available fromCelltech, Ltd., Berkshire, UK) was cut with the BamH1 restriction enzymeand dephosphorylated with calf intestinal alkaline phosphatase (CIAP) toprevent the vector from religating to itself. The BamH1 cut pSVLGS.1fragment was then ligated to a 2.4 kb BamH1 to Bgl2 fragment of pEE6hCMV(described in PCT Application No. WO89/01036, also available fromCelltech) which was cut with Bgl2, BamH1 and Fsp1 to avoid two fragmentsof similar size, to yield an 11.2 kb vector designated p6/PSVLGS.1.pSVLGS.1 contains the glutamine synthetase selectable marker gene undercontrol of the SV40 later promoter. The BamH1 to Bgl2 fragment ofpEE6hCMV contains the human cytomegalovirus major immediate earlypromoter (hCMV), a polylinker, and the SV40 early polyadenylationsignal. The coding sequences for soluble TNF-R were added to p6/PSVLGS.1by excising a Not1 to BamH1 fragment from the expression vectorpsolTNFR/CAVNOT (made according to Example 3 above), blunt ending withKlenow and ligating with Sma1 cut dephosphorylated p6/PSVLGS.1, therebyplacing the solTNF-R coding sequences under the control of the hCMVpromoter. This resulted in a single plasmid vector in which the SV40/GSand hCMB/solTNF-R transcription units are transcribed in oppositedirections. This vector was designated psolTNFR/P6/PSVLGS.

psolTNFR/P6/PSVLGS was used to transfect CHO-K1 cells (available fromATCC, Rochville, Md., under accession number CCL 61) as follows. Amonolayer of CHO-K1 cells were grown to subconfluency in MinimumEssential Medium (MEM) 10× (Gibco: 330-1581AJ) without glutamine andsupplemented with 10% dialysed fetal bovine serum (Gibco: 220-6300AJ), 1mM sodium pyruvate (Sigma), MEM non-essential amino acids (Gibco:320-1140AG), 500 μM asparagine and glutamate (Sigma) and nucleosides (30μM adenosine, guanosine, cytidine and uridine and 10 μMthymidine)(Sigma).

Approximately 1×10⁶ cells per 10 cm petri dish were transfected with 10μg of psolTNFR/P6/PSVLGS by standard calcium phosphate precipitation,substantially as described by Graham & van der Eb, Virology 52: 456(1983). Cells were subjected to glycerol shock (15% glycerol inserum-free culture medium for approximately 1.5 minutes) approximately 4hours after transfection, substantially as described by Frost &Williams, Virology 91: 39 (1978), and then washed with serum-freemedium. One day later, transfected cells were fed with fresh selectivemedium containing MSX at a final concentration of 25 μM. Colonies ofMSX-resistant surviving cells were visible within 3-4 weeks. Survivingcolonies were transferred to 24-well plates and allowed to grow toconfluency in selective medium. Conditioned medium from confluent wellswere then assayed for soluble TNF-R activity using the binding assaydescribed in Example 1 above. These assays indicated that the coloniesexpressed biologically active soluble TNF-R.

In order to select for GS gene amplification, several MSX-resistant celllines are transfected with psolTNFR/P6/PSVLGS and grown in variousconcentrations of MSX. For each cell line, approximately 1×10⁶ cells areplated in gradually increasing concentrations of 100 μM, 250 μM, 500 μMand 1 mM MSX and incubated for 10-14 days. After 12 days, coloniesresistant to the higher levels of MSX appear. The surviving colonies areassayed for TNF-R activity using the binding assay described above inExample 1. Each of these highly resistant cell lines contains cellswhich arise from multiple independent amplification events. From thesecells lines, one or more of the most highly resistant cells lines areisolated. The amplified cells with high production rates are then clonedby limiting dilution cloning. Mass cell cultures of the transfectantssecrete active soluble TNF-R.

Example 8 Expression of Soluble Human TNF-R in Yeast

Soluble human TNF-R was expressed in yeast with the expression vectorpIXY432, which was derived from the yeast expression vector pIXY120 andplasmid pYEP352. pIXY120 is identical to pYαHuGM (ATCC 53157), exceptthat it contains no cDNA insert and includes a polylinker/multiplecloning site with a Nco1 restriction site.

A DNA fragment encoding TNF receptor and suitable for cloning into theyeast expression vector pIXY120 was first generated by polymerase chainreaction (PCR) amplification of the extracellular portion of the fulllength receptor from pCAV/NOT-TNF-R (ATCC 68088). The following primers(encoding amino acids corresponding in part to Leu¹-Thr⁸ andPro²²⁵-Asp²³⁵ of SEQ ID NO:1) were used in this PCR amplification:

5′ End Primer 5′-TTCCGGTACCTTTGGATAAAAGAGACTACAAGGACAsp718- >ProLeuAspLysArgAspTyrLysAsp (SEQ ID NO: 18)GACGATGACAAGTTGCCCGCCCAGGTGGCATTTACA-3′ (SEQ ID NO: 19)AspAspAspLys<--------TNF-R----------> 3′ End Primer (antisense) (SEQ IDNO 20) 5′-CCCGGGATCCTTAGTCGCCAGTGCTCCCTTCAGCTGGG-3′       BamH1>End<-------------TNF-R------->The 5′ end oligonucleotide primer used in the amplification included anAsp718 restriction site at its 5′ end, followed by nucleotides encodingthe 3′ end of the yeast α-factor leader sequence (Pro-Leu-Asp-Lys-Arg(SEQ ID NO:21)) and those encoding the 8 amino acids of the FLAG®peptide (AspTyrLysAspAspAspAspLys (SEQ ID NO:22)) fused to a sequenceencoding the 5′ end of the mature receptor. The FLAG® peptide (Hopp etal., Bio/Technology 6: 1204, 1988) is a highly antigenic sequence whichreversibly binds the monoclonal antibody M1 (ATCC HB 9259). Theoligonucleotide used to generate the 3′ end of the PCR-derived fragmentis the antisense strand of DNA encoding sequences which terminate theopen reading frame of the receptor after nucleotide 704 of the maturecoding region (following the Asp residue preceding the transmembranedomain) by introducing a TAA stop codon (underlined). The stop codon isthen followed by a BamH1 restriction site. The DNA sequences encodingTNF-R are then amplified by PCR, substantially as described by Innis etal., eds., PCR Protocols: A Guide to Methods and Applications (AcademicPress, 1990).

The PCR-derived DNA fragment encoding soluble human TNF-R was subclonedinto the yeast expression vector pIXY120 by digesting the PCR-derivedDNA fragment with BamH1 and Asp718 restriction enzymes, digestingpIXY120 with BamH1 and Asp718, and ligating the PCR fragment into thecut vector in vitro with T4 DNA ligase. The resulting construction(pIXY424) fused the open reading frame of the FLAG®-soluble TNF receptorin-frame to the complete α-factor leader sequence and placed expressionin yeast under the aegis of the regulated yeast alcohol dehydrogenase(ADH2) promoter. Identity of the nucleotide sequence of the soluble TNFreceptor carried in pIXY424 with those in cDNA clone 1 were verified byDNA sequencing using the dideoxynucleotide chain termination method.pIXY424 was then transformed into E. coli strain RR1.

Soluble human TNF receptor was also expressed and secreted in yeast in asecond vector. This second vector was generated by recovering thepIXY424 plasmid from E. coli and digesting with EcoR1 and BamH1restriction enzymes to isolate the fragment spanning the region encodingthe ADH2 promoter, the α-factor leader, the FLAG®-soluble TNF receptorand the stop codon. This fragment was ligated in vitro into EcoR1 andBamH1 cut plasmid pYEP352 (Hill et al., Yeast 2: 163 (1986)), to yieldthe expression plasmid pIXY432, which was transformed into E. colistrain RR1.

To assess secretion of the soluble human TNF receptor from yeast,pIXY424 was purified and introduced into a diploid yeast strain of S.cerevisiae (XV2181) by electroporation and selection for acquisition ofthe plasmid-borne yeast TRP1⁺ gene on media lacking tryptophan. Toassess secretion of the receptor directed by pIXY432, the plasmid wasintroduced into the yeast strain PB149-6b by electroporation followed byselection for the plasmid-borne URA3⁺ gene with growth on media lackinguracil. Overnight cultures were grown at 30° C. in the appropriateselective media. The PB149-6b/pIXY434 transformants were diluted intoYEP-1% glucose media and grown at 30° C. for 38-40 hours. Supernatantswere prepared by removal of cells by centrifugation, and filtration ofsupernatants through 0.45μ filters.

The level of secreted receptor in the supernatants was determined byimmuno-dotblot. Briefly, 1 ul of supernatants, and dilutions of thesupernatants, were spotted onto nitrocellulose filters and allowed todry. After blocking non-specific protein binding with a 3% BSA solution,the filters were incubated with diluted M1 anti-FLAG® antibody, excessantibody was removed by washing and then dilutions of horseradishperoxidase conjugated anti-mouse IgG antibodies were incubated with thefilters. After removal of excess secondary antibodies, peroxidasesubstrates were added and color development was allowed to proceed forapproximately 10 minutes prior to removal of the substrate solution.

The anti-FLAG® reactive material found in the supernatants demonstratedthat significant levels of receptor were secreted by both expressionsystems. Comparisons demonstrated that the pIXY432 system secretedapproximately 8-16 times more soluble human TNF receptor than thepIXY424 system. The supernatants were assayed for soluble TNF-Ractivity, as described in Example 1, by their ability to bind ¹²⁵I-TNFαand block TNFα binding. The pIXY432 supernatants were found to containsignificant levels of active soluble TNF-R.

Example 9 Isolation of Murine TNF-R cDNAs

Murine TNF-R cDNAs were isolated from a cDNA library made from murine7B9 cells, an antigen-dependent helper T cell line derived from C57BL/6mice, by cross-species hybridization with a human TNF-R probe. The cDNAlibrary was constructed in λZAP (Stratagene, San Diego), substantiallyas described above in Example 2, by isolating polyadenylated RNA fromthe 7B9 cells.

A double-stranded human TNF-R cDNA probe was produced by excising anapproximately 3.5 kb Not1 fragment of the human TNF-R clone 1 and³²P-labeling the cDNA using random primers (Boehringer-Mannheim).

The murine cDNA library was amplified once and a total of 900,000plaques were screened, substantially as described in Example 2, with thehuman TNF-R cDNA probe. Approximately 21 positive plaques were purified,and the Bluescript plasmids containing EcoR1-linkered inserts wereexcised (Stratagene, San Diego). Nucleic acid sequencing of a portion ofmurine TNF-R clone 11 indicated that the coding sequence of the murineTNF-R was approximately 88% homologous to the corresponding nucleotidesequence of human TNF-R. A partial nucleotide sequence of murine TNF-RcDNA clone 11 is set forth in SEQ ID NO:3.

Example 10 Preparation of Monoclonal Antibodies to TNF-R

Preparations of purified recombinant TNF-R, for example, human TNF-R, ortransfected COS cells expressing high levels of TNF-R are employed togenerate monoclonal antibodies against TNF-R using conventionaltechniques, for example, those disclosed in U.S. Pat. No. 4,411,993.Such antibodies are likely to be useful in interfering with TNF bindingto TNF receptors, for example, in ameliorating toxic or other undesiredeffects of TNF, or as components of diagnostic or research assays forTNF or soluble TNF receptor.

To immunize mice, TNF-R immunogen is emulsified in complete Freund'sadjuvant and injected in amounts ranging from 10-100 μg subcutaneouslyinto Balb/c mice. Ten to twelve days later, the immunized animals areboosted with additional immunogen emulsified in incomplete Freund'sadjuvant and periodically boosted thereafter on a weekly to biweeklyimmunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot-blotassay (antibody sandwich) or ELISA (enzyme-linked immunosorbent assay).Other assay procedures are also suitable. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to the murine myelomacell line NS1. Hybridoma cell lines generated by this procedure areplated in multiple microtiter plates in a HAT selective medium(hypoxanthine, aminopterin, and thymidine) to inhibit proliferation ofnon-fused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith TNF R, for example, by adaptations of the techniques disclosed byEngvall et al., Immunochem. 8: 871 (1971) and in U.S. Pat. No.4,703,004. Positive clones are then injected into the peritonealcavities of syngeneic Balb/c mice to produce ascites containing highconcentrations (>1 mg/ml) of anti-TNF-R monoclonal antibody. Theresulting monoclonal antibody can be purified by ammonium sulfateprecipitation followed by gel exclusion chromatography, and/or affinitychromatography based on binding of antibody to Protein A ofStaphylococcus aureus.

DETAILED DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 and SEQ ID NO:2 show the partial cDNA sequence and derivedamino acid sequence of the human TNF-R clone 1. Nucleotides are numberedfrom the beginning of the 5′ untranslated region. Amino acids arenumbered from the beginning of the signal peptide sequence. The putativesignal sequence is represented by amino acids −22 to −1. The N-terminusof the mature TNF-R begins with amino acid 1. The predictedtransmembrane region extends from amino acids 236-265.

SEQ ID NO:3 and SEQ ID NO:4 show the cDNA sequence and derived aminoacid sequence of the murine TNF-R clone 11. The putative signal peptidesequence is represented by amino acids −22 to −1. The N-terminus of themature TNF-R begins with amino acid 1. The predicted transmembraneregion extends from amino acids 234-265.

1. A purified preparation of a protein, wherein the protein comprisesamino acids 1-163 of SEQ ID NO:2, wherein the protein has been purifiedby a method comprising the following steps: (a) concentrating asupernatant from a mammalian cell culture which contains the protein;(b) purifying the protein in the concentrated supernatant by subjectingit to affinity chromatography and/or ion exchange chromatography.
 2. Thepurified preparation of claim 1, wherein the protein comprises aminoacids 1-185 of SEQ ID NO:2.
 3. The purified preparation of claim 2,wherein the protein comprises amino acids 1-235 of SEQ ID NO:2.
 4. Thepurified preparation of claim 1, wherein the mammalian cell culture is aCHO cell culture.
 5. The purified preparation of claim 1, wherein theprotein has been subjected to affinity chromatography and ion exchangechromatography.
 6. The purified preparation of claim 5, wherein ionexchange chromatography is done after affinity chromatography.
 7. Thepurified preparation of claim 5, wherein the ion exchange chromatographyis anion exchange chromatography.
 8. The purified preparation of claim7, wherein the anion exchange chromatography is performed on a matrixhaving pendant diethylaminoethyl (DEAE) groups.
 9. The purifiedpreparation of claim 1, wherein the ion exchange chromatography is anionexchange chromatography.
 10. The purified preparation of claim 9,wherein the anion exchange chromatography is performed on a matrixhaving pendant diethylaminoethyl (DEAE) groups.
 11. The purifiedpreparation of claim 1, wherein the ion exchange chromatography iscation exchange chromatography.
 12. The purified preparation of claim11, wherein the cation exchange chromatography is performed on a matrixhaving pendant carboxymethyl or sulfopropyl groups.
 13. The purifiedpreparation of claim 1, wherein the purified preparation comprises lessthan about one percent by weight of non-human cell components.
 14. Apurified preparation of a protein comprising less than about one percentby weight of non-human cell components, wherein the protein comprisesamino acids 1-163 of SEQ ID NO:2.
 15. The purified preparation of claim14, wherein the protein has been purified from a supernatant of amammalian cell culture.
 16. The purified preparation of claim 15,wherein the protein has been purified by a method comprising thefollowing steps: (a) concentrating the supernatant; (b) purifying theprotein in the concentrated supernatant by subjecting it to affinitychromatography.
 17. The purified preparation of claim 15, wherein theprotein has been purified by a method comprising the following steps:(a) concentrating the supernatant; (b) purifying the protein in theconcentrated supernatant by subjecting it to ion exchangechromatography.
 18. The purified preparation of claim 17, wherein theion exchange chromatography is anion exchange chromatography.
 19. Thepurified preparation of claim 17, wherein the ion exchangechromatography is cation exchange chromatography.
 20. The purifiedpreparation of claim 15, wherein the mammalian cell culture is a CHOcell culture.